System for releasing and isolating nucleic acids

ABSTRACT

An instrument that can hold one or more sample processsing vessels, maintain the sample processing vessels at a constant temperature, shake the sample processing vessels and separate magnetic particles by means of magnetic force. This system greatly simplifies the isolation of nucleic acids.

The object of the invention is a system for the release and isolation ofnucleic acids and a procedure for using this system.

Detection procedures based on the determination of nucleic acids in asample have increased in significance recently. This is due, among otherthings, to the high sensitivity of detection that these procedures canachieve. In terms of sensitivity, nucleic acid detection procedures arebasically superior to antigen detection procedures. Although antigensare often relatively accessible in a sample, numerous steps are usuallyrequired to make nucleic acids accessible, especially when detectingorganisms. In addition, nucleic acids are usually present in very lowconcentrations. Purification procedures for isolating nucleic acids fromsamples containing cells are known in particular, although they requirea great deal of time and effort.

The sensitivity of the sample enrichment and pretreatment systems fornucleic acids currently on the market is often insufficient. Inaddition, automated sample pretreatment systems are not sufficientlysafe from contamination to enable amplification, e.g. using the PCR.Another disadvantage of the automated sample pretreatment systemscurrently available is that they require the use of organic solvents(phenol and/or chloroform alcohol mixtures) to extract the nucleicacids.

The procedures in use today that immobilize nucleic acids basically usetwo principles to isolate nucleic acids. One principle calls for liquidsamples containing nucleic acids to be aspirated through a solid phasewhich retains the nucleic acids. This step is preceded by a lysis stepperformed in a separate container. The nucleic acids are then dissolvedfrom the solid matrix by aspirating an elution fluid through the matrix.The elation solution containing the nucleic acids is aspirated into acontainer for the next steps. It has been demonstrated, however, thatthe purity of the devices in use today does not meet the requirementsfor a subsequent amplification reaction, such as the PCR.

According to the second principle of nucleic acid isolation, the nucleicacids are removed by way of precipitation and then separated in acentrifuge. This procedure cannot be performed in a "batch" mode,however. Rather, it first requires that a solution containing cells betreated with lysing agents in a reaction vessel. The reaction mixture isthen transferred by pipette from the container to a centrifugation tube.This tube contains an insert to which the released nucleic acids canadsorb, while the remaining fluid can flow to the bottom of the tubeduring centrifugation. The insert is treated one or more times with afluid to wash the absorbed nucleic acids. For this step, the insert istransferred to a second centrifugation tube so that residues from thesample fluid do not reenter the insert. In the final step, the insert isplaced in yet another container. An elution solution is then centrifugedthrough the insert to transfer the nucleic acids to another vessel thatcontains a solution that is capable of being processed further. Thisprocedure is very susceptible to contamination, however, and requirestransferring solutions between numerous reaction vessels.

The task of this invention was to provide a system for which thedisadvantages of the state of the art are eliminated either completelyor at least partially. In particular, this system can be used to absorband desorb nucleic acids to a solid phase matrix without requiring acentrifuge for these steps.

A main feature of the invention is a receptacle for a sample processingvessel that can be kept at a constant temperature and set into motion inorder to thoroughly mix the substances contained in the sampleprocessing vessel. In addition, this receptacle is connected to avacuum-generating system (e.g. a hose pump or a piston pump). Thereceptacle also makes it possible to separate magnetic particles withinthe sample processing vessel. Important advantages of the invention arethe protection it provides against contamination (between samples andbetween the system and the environment), and its potential to holdenough sample processing vessels to ensure cost effectiveness.

This invention also provides a procedure for releasing and isolating ordetecting nucleic acids from biological compartments of a sample withthe following steps:

The sample is incubated in a sample processing vessel with magneticparticles that can bind with the biological compartments while thesample processing vessel is shaken,

A magnet is positioned near the sample processing vessel in order tohold the magnetic particles against the wall of the vessel,

The remaining fluid is removed from the sample processing vessel,

The magnetic particles are resuspended in a second fluid by

a) removing the magnet away from the sample processing vessel so thatthe magnetic particles are no longer held against the wall of thevessel, while

b) shaking the sample processing vessel,

The biological compartments are warmed and lysed,

The lysis mixture is cooled under conditions that make it possible toimmobilize or hybridize the nucleic acids to be isolated or detected.

This invention also provides a procedure for the release and isolationof nucleic acids from a suspension of biological compartments usingmagnetic particles with the following steps:

A sample is incubated in a sample processing vessel with magneticparticles in order to lyse the biological compartments,

The lysis mixture is cooled and the nucleic acids to be isolated ordetected are immobilized on the magnetic particles,

The state of immobilization is eliminated and the nucleic acids to beisolated and purified are transferred to a vessel from which they can bepipetted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 and 2 show a system for the release and/or isolation of nucleicacids in accordance with the invention.

FIG. 3 shows a procedure for isolating nucleic acids provided in theinvention.

FIGS. 4, 5, 6, 7 and 8 show another construction of a system for therelease and isolation of nucleic acids in accordance with the invention.

Nucleic acids" as described in context with this invention refer tonucleic acids that are present in biological compartments. "Biologicalcompartments" refer in particular to viruses or cells of bacterialorigin for instance. In an especially preferred construction, the cellsare basically present in a format in which they are basically separatefrom each other. In principle, however, this invention can also processmulticellular compartments. These compartments and their nucleic acidsare contained in a sample at the beginning of the procedure described bythis invention. This sample is preferably a suspension of biologicalcompartments in a fluid. These fluids can be obtained from bodily fluidssuch as blood, saliva or urine, for instance.

"Release of nucleic acids" as described in context with this inventionrefers to the discharge of nucleic acids from the biologicalcompartments. Any means can be used to discharge the nucleic acids fromthe biological compartments. Preferably, the wall separating thebiological compartments from the fluid is destroyed. This can beaccomplished, for instance, by treating the compartments with cell walldestroying agents such as proteinase K. "Isolation of nucleic acids" asdescribed in context with this invention refers to the separation ofnucleic acids from other components in the sample. "Other components inthe sample" can include the walls of the biological compartments, theirdegradation products, other contents of the biological compartments andcomponents of the fluid that surrounds the biological compartments inthe sample. These components can include proteins or enzyme inhibitors,especially enzymes that degrade nucleic acids, such as Dnase or RNase.In this sense, isolation can also refer to a method for purify nucleicacids. This isolation step can be specific or non-specific for othernucleic acids contained in the sample.

"Detection of nucleic acids" as described in context with this inventionrefers to a procedure in which the presence or quantity of nucleic acidsis determined. These procedures can be performed quantitatively orqualitatively. The quantitative detection procedure usually requiresthat a comparison test be performed with a sample that contains a knownquantity of the nucleic acids to be detected. The detection can besequence-specific or sequence non-specific. To make the detectionspecific, one usually uses "probes" that are characterized by the factthat they have a nucleobase sequence that is more or less characteristicfor the nucleic acids in the sample. If the goal is to perform specificdetection of nucleic acids, a probe is used that contains a basesequence that is complementary to the base sequence of the nucleic acidsto be detected, but not, however, to other nucleic acids in the sample.Probes can be molecules that contain a group that can be detected eitherdirectly or indirectly. Groups that can be detected directly includeradioactive (³² P), colored or fluorescent groups or metal atoms. Groupsthat can be detected indirectly include, for instance, compounds with animmunological or enzymatic effect such as antibodies, antigens, haptens,enzymes or enzymatically active sub-enzymes. These groups are detectedin a subsequent reaction or sequence of reactions. Especially preferredare haptens, such as digoxigenin or biotin. Hapten-labelled probes ofthis nature can then be detected easily in a reaction with a labelledantibody against the hapten.

DESCRIPTION OF THE SYSTEM

The object of the invention is a system for the release and/or isolationof nucleic acids from a suspension of biological compartments comprisingthe following components:

a receptacle (10) for one or more sample processing vessels (A),

a thermostat unit (20) to maintain the sample processing vessels (A) andthe fluids they contain at a constant temperature,

a mechanical shaker (30) to shake the sample processing vessels (A),

a separation device (40) to separate magnetic particles from fluid usingmagnetic force and deposit them on a wall of each sample processingvessel (A), and

a pump unit (50) to remove fluid from the sample processing vessel (A).

The surface of the system is easy to clean and protects the operatorfrom burns (e.g. by virtue of a plastic housing).

Drawings of a system comprising the units described by this inventionare shown in FIGS. 1 and 2.

The system has a receptacle (10) for holding the sample processingvessels. This receptacle contains numerous recesses (12) or "cavities"into which the sample vessels are placed. The cavities are preferablyarranged in a linear format or in subgroups that are arranged in alinear format yet are at right angles to each other. The distancebetween the cavities is preferably the same distance that separates thewells of a microtiter plate and, more preferably, twice this distance.The cavities are designed to hold the sample vessels.

The sample processing vessel (A) can basically have any shape or design.For instance, these sample processing vessels can be the wells of amicrotiter plate (e.g. a 96-well format). The vessels are preferablycylindrical, however, with an opening at the top to receive fluid and,most preferably, an opening at the bottom from which fluid can exit(A11). A sample processing vessel with this design can be used to reducecontamination while processing samples containing nucleic acids. Thesevessels are usually made of a plastic such as polypropylene.

In an especially preferred construction of this invention, vessels canbe used that are described in the German design patent applicationsnumbered 29505652.5 and 29505707.6.

The fluid in the sample processing vessels is kept at a constanttemperature in the system described by this invention by means of athermostat unit (20). This unit, which basically consists of componentscommon to thermostats, is preferably integrated--at least partially--inthe receptacle (10) in which the sample processing vessels can beplaced. The receptacle (10) especially includes a metal block with goodthermal conductivity. The shape of this metal block is designed to fitwith the outer contour of the sample processing vessels in such a waythat the walls of the sample processing vessels placed in the receptaclefit within the metal block as snugly as possible, so that heat can betransferred efficiently. The temperature in the block is increased ordecreased, depending on the reaction to be performed in the sampleprocessing vessel. The temperature can range from 4° C. to 95° C.Ideally, numerous electrical heating elements located as closely aspossible to the sample processing vessel are used to increase thetemperature. The fluid in the sample processing vessel is cooled bymeans of peltier elements that are also located as close to the sampleprocessing vessels as possible. The temperature is regulated preferablyby means of a fuzzy regulator or a PIDT regulator. A sufficient numberof heat sensors are situated in proper locations within the thermostatunit.

Another construction of the thermostat unit (20) includes a blockthrough which fluid flows. Fluids--usually water or aqueous salinesolutions--are used as the incubation medium. Preferably, the incubationfluid is fed directly into openings (21) in the block (10) by means of acirculating pump via flexible tubes connected to a heating or coolingsystem. The volume of the incubation reservoir (represented in FIG. 1with "heating unit" and "cooling unit") outside the DNA module is muchlarger than the dead volume of the DNA module in order to minimize thisdisturbance variable when the 2-way valve is switched over. The heatingand cooling units maintain the fluid at a constant temperature beforethe process begins and can be programmed to operate at certain intervalsif necessary. A regulator controls the valve and heating and coolingunits, and, coupled with the appropriate volume flow achieved by thecirculating pump, maintains the heating and cooling rates desired. The"thermostat unit" (20) in this construction refers to a combination of ablock, circulating pump, heating unit, cooling unit and 2-way valvethrough which fluid flows. The "DNA module" refers to the device thatincludes the receptacle for the sample processing vessel, the shakingdevice and the separation device.

The system enables one to work with magnetic particles (beads). The term"magnetic particles" refers to particles that can be transported in acertain direction by means of magnetic force. These particles can bemade of ferromagnetic or superparamagnetic materials, for instance.Especially preferred in the context of this invention are ferromagneticmaterials. Particles are solid materials with a small diameter. Withinthe context of this invention, particles with an average particle sizeof more than 2.8 μm but less than 200 μm are especially suitable. Mostpreferably they have an average particle size of between 10 and 15 μm.The distribution of particle size is preferably homogenous. The surfaceof these particles is modified in such a way that they can bind with thebiological compartments. Magnetic particles that are suitable for thisapplication are the known and commercially available latex magneticparticles to which antibodies can be bound, for instance. Antibodiestargeted against surface antigens of the biological compartments areused in particular to bind the biological compartments to the magneticparticles. Magnetic particles of this nature are also commerciallyavailable.

Glass-magnetic pigments with surfaces to which nucleic acids can bindcan also be used. Glass-magnetic pigments of this nature are known fromthe German patent application number 19537985.3.

For the procedure described by this invention to be performedsuccessfully, the magnetic particles must be bound to the inner wall ofthe sample processing vessel for certain reaction steps and then broughtback into suspension in a subsequent step. In an especially preferredconstruction of this invention, a separation device (40) to which one ormore permanent magnets or electromagnets are attached is moved towardsthe sample processing vessel in order to position the magnets. Theremoval of the magnet away from the sample processing container--whichis necessary for separation to be performed efficiently--depends to alarge extent on the strength of the magnetic field that the magnets canproduce, the size of the magnetic particles, and on the ability of themagnetic particles to be magnetized. The nature of the subsequentprocessing steps (e.g. mechanical stressing of the magnets) alsodetermines the strength of the magnetic field to be used. If a permanentmagnet is used, it is moved from a position where it cannot separate themagnetic particles and into the vicinity of the vessel so that themagnetic particles are held against the vessel wall. If anelectromagnetic is used, it is turned on and allowed to remain in thepower ON state until the biological compartments held against the vesselwall are processed. "Positioning a magnet in the vicinity of the vessel"also refers to the case in which the vessel is brought into the vicinityof the magnet Ultimately, it refers merely to the motion of the magnetin relation to the vessel. The separation device (40) preferably has amagnet that can be moved towards the sample processing vessel along apredetermined path, e.g. on rails or, preferably, by moving the magneton a circular track, e.g. along an axis that passes next to the samplevessel. The separation unit also comprises a motor that can drive themovement of the magnet towards and away from the sample processingvessel.

In another construction, the separation unit (40) has a gear rack thatcan be driven in linear fashion by means of a d.c. motor or a steppingmotor. A device that holds the magnets--permanent magnets in thiscase--is positioned at a right angle to the gear rack. Light barrierscan be used to detect the end positions of the movement of the magnets.In the preferred construction, one magnet is assigned to each sampleprocessing vessel, the front face of which is moved into position nextto the vessel. Two magnets can also be moved into position next to eachsample processing vessel, however. Preferably, one magnet is moved intoposition next to 2 vessels, so that only n+1 magnets are required for nvessels.

For the separation step, the magnets must be moved as close to thesample processing vessels as possible in order to achieve a high rate ofseparation of the magnetic particles. It is also important that thedistance traveled by the magnets between the positions be sufficientlylong. Depending on the material and geometry of the magnet used, themagnets must be up to 40 mm away from the sample processing vessel toprevent unintentional separation of the magnetic particles in the sampleprocessing vessel. For the instrument to function properly, it isimportant that the magnetic forces only affect the particles in thesample processing vessel, if this is so desired. To achieve this effect,the inactive position of the magnet must be far enough away from thevessel that the magnetic field has no effect on the movement of theparticles.

This effect can also be achieved in such a way that the distance betweenthe magnet and the sample processing vessel is not changed. The magneticfields can simply be interrupted by a μ-metal that is moved into alocation between the vessel and the magnet.

In another possible construction, the magnets are arranged on arotatable shaft driven by a d.c. motor. This construction enables themagnets to be moved along a circular path. The magnets themselves can bearranged on this shaft in any order that moves them towards or away fromthe sample processing vessels. In addition, a drive mechanism ispreferred in which one shaft with a number of magnets is situated oneach opposing side of the system. The shafts are driven by a motor bymeans of a gear rack. With this arrangement, every two magnets move in asynchronous motion towards the front face of the sample processingvessel.

Magnets for the invention described here preferably have a mass ofbetween 0.5 and 5 g, and, especially preferred, between 1 and 4 g. Theouter dimensions of the prototype are 10 mm×10 mm×3 mm. Materials thathave been proven to be suitable for a permanent magnet are rare earthmaterials (e.g. NeFeB, VACODYM 370 HR) with an optimal BH maximum at thesmallest dimensions. For the separation step to proceed efficiently, itis advantageous to design the gradients of the magnetic field to beespecially pronounced. For this reason, the magnets should be located asclose to the vessel as possible. The sample processing vessels arepreferably made of materials that weaken the magnetic field as little aspossible, such as polypropylene.

Tubes (51) that are connected to a vacuum-generating system are attachedto the underside of the cavity on the unit that holds the sampleprocessing vessels. One tube is assigned to each cavity. Since there areopenings in the bottom of the sample processing vessels, a vacuum can becreated to aspirate the contents of the sample processing vessels anddeposit them in the waste container. In the construction shown in FIG.2, each cavity has a seal that prevents air from being introduced intothe space between the sample processing vessel and the inlet (14) whenthe waste material is aspirated.

The vacuum system basically comprises a piston pump (50) that isconnected with the cavities via a tubing system. A waste container intowhich the fluid is deposited is located between the piston pump and thecavities. The fluid that is aspirated out of the sample processingvessels is waste. In addition, a valve is situated between every sampleprocessing vessel and the waste container. This valve enables the vacuumto be switched to each cavity when the system is permanently evacuatedfrom the pump, past the waste container, and up to the valve. With theconstruction provided by the invention, the sample processing vesselscan be aspirated in parallel and sequentially.

In another construction, piston pumps and valves are replaced with aflow inducer. The waste container is not evacuated permanently in thiscase, rather, it is situated in the fluid stream behind the cavities andthe flow inducer. With this arrangement, the cavities can only beaspirated in parallel. Numerous flow inducers may be used as well, whichthen serve a certain number of cavities and enable work to be performedin a partially sequential fashion.

The sample processing vessels are preferably moved in a horizontaldirection. In an especially preferred construction, the receptacle(10)--which contains recesses (12) that hold one sample vessel each--ismoved, so that all sample vessels in the system are shaken. Vibrationabsorbers (11) serve to reduce the amount of movement transferred to therest of the instrument. This invention preferably uses a mechanicalshaker (30) that moves the sample processing vessels (A). This unit canbasically be any mechanical device that is suitable for mixing fluids ina vessel. A preferred example of such a unit is described below.

A stepping motor with an eccentric cam and an equalizing weight situatedon a fixed framework (1) move the receptacle--which is placed onvibration absorbers on this framework--in a circular, eccentric pathwith a fixed amplitude and a variable frequency. The preferred amplitudeA is ≦1.5 mm, and the preferred frequency (f) is greater than 1 Hz andless than 50 Hz. The mixing and resuspension step lasts between 5 and 30s, depending on the physical characteristics of the sample material. Theamplitude can be varied by replacing the eccentric cam.

Combining the system provided by this invention with an automatedpipetting system is not a logical step, because this would require thatthe sample vessels be placed in a definite position before and duringthe pipetting steps. If the sample vessels are not placed in a definiteposition, they are located in a different position after the shakingstep. If the position of the vessels during the pipetting step is notspecified exactly, it may not be possible to perform the pipettingprocedure correctly. For this reason, the instrument ensures that thevessel is located in a defined "home" position after the shakingprocedure, from where a pipetting step or other processes can beperformed.

It is advantageous to use a stepping motor instead of a d.c. motor inorder to ensure a defined home position. In a preferred construction,the home position is detected with a light barrier.

The instrument may also be designed to be non-invasive, as describedbelow. However, these alternatives are more complex in design (Versions1 and 2), or they utilize mixing steps (3) that take a longer time tocomplete:

1. A combination of one, two or three linear drives for the receptacleon the plane or in a space (X, Y and Z-axis) to create Lissajous curves,for instance.

2. Wobbling by tilting the framework (1) at a certain angle and placingthe receptacle (10) at the opposite end.

3. Magnetic stirring mechanism

4. Swirling or tapping the DNA module.

The system components are coupled in such a way as to be functional,e.g. via integration of the magnets in the unit (10). The systemcomponents are also coupled chronologically. For instance, the units areoperated in the sequence required for the application desired, e.g. viaa computer program or by the operator initiating the steps individually.

Description of the Procedure Version A

In the initial step, the sample is incubated in a sample processingvessel with magnetic particles (beads) that can bind with the biologicalcompartments while the sample processing vessel is shaken.

The incubation of the samples with the magnetic particles can take placein any fashion. It is necessary for the sample and the magneticparticles to be placed in the sample processing vessel. Neither themethod by which the sample and magnetic particles are placed in thevessel nor the sequence in which this takes place is especiallysignificant for the procedure provided by this invention. Preferably,however, the magnetic particles are pipetted into the sample processingvessel in a suspension with a known concentration of magnetic particles.The sample is pipetted into the sample processing vessel either beforeor after the suspension of magnetic particles.

The mixture is incubated under appropriate conditions until a sufficientquantity of biological compartments are bound to the magnetic particles,usually between 1 minute and 10 minutes. The sample processing vessel ispreferably closed in proper fashion, e.g. with a cap and/or a valve.

An important feature of the invention is the fact that the mixture inthe sample processing vessel is shaken during incubation. The mixturecan be shaken in intervals, or it can be shaking during the entireincubation period or only during certain periods. The mixture is shakenin order to sufficiently mix the biological compartments and themagnetic particles in the fluid, and especially to suspend or resuspendthe beads and accelerate diffusion. This reduces the amount of timerequired to bind the biological compartments to the magnetic particles.

After the incubation step and after the compartments are bound to themagnetic particles, the biological compartments are removed from thesurrounding fluid in the sample. An appropriate method of accomplishingthis is to collect the magnetic particles and bound biologicalcompartments by positioning a magnet near the sample processing vessel.This holds the magnetic particles with the biological compartmentsagainst the vessel wall, as is preferable. The beads are thereforeusually separated on the inside wall of the sample processing vessel ora part thereof that is located below the surface of the sample fluid.

The fluid surrounding the biological compartments is then removed fromthe sample processing vessel. This is performed under conditions inwhich the magnetic particles remain against the vessel wall. The methodused to remove the sample depends on the type of sample processingvessel used. The fluid can be removed via pipette, for instance. In apreferred construction, however, in which the sample processing vesselhas an opening at the bottom from which the fluid can exit, the fluid issimply aspirated through this opening. This fluid removal methodminimizes the mechanical stress placed on the magnetic particles andthereby prevents them from being removed from the vessel wall.

An especially important step is the resuspension of the magneticparticles that remain on the vessel wall in a second fluid that isadded. To accomplish this, the magnet is moved away from the vessel sothat it no longer holds the magnetic particles against the vessel wall.As described above, it is also possible to move the vessel away from themagnet. With regard for the invention described here, it has beendemonstrated that simply removing the magnet is not enough to resuspendthe magnetic particles in the solution if the vessel is not also shaken,and preferably simultaneously. This shaking motion is performed by themechanical shaker (30). It causes the magnetic particles to bedistributed evenly within the second fluid. This second fluid can beadded to the sample processing vessel, e.g. via pipette, before or afterthe magnet is removed.

The procedure provided by this invention can also be used to furtherpurify biological compartments. To accomplish this, a suspension ofmagnetic particles that bind with the biological compartments ispositioned in a sample processing vessel in relation to a magnet in sucha way that the magnetic particles with the biological compartments areheld against the vessel wall. The fluid that contains the biologicalcompartments is then removed from the vessel and the magnetic particlesare resuspended in a second fluid--a wash fluid in this case--by movingthe magnet away from the vessel so that the magnetic particles are nolonger held against the vessel wall, while the vessel is shaken. Thiswash step can be repeated as needed until the biological compartmentshave reached a sufficient level of purity.

Another step of the procedure provided by this invention is thesubsequent disintegration (lysis) of the biological compartments.Procedures for lysing biological compartments are known by the expert,as are the specific conditions for certain types of compartments, e.g.cells. To lyse bacteria, for instance, a mixture of proteinase K isadded to the biological compartments and incubated for the amount oftime necessary to lyse or partially or completely decompose the cellwalls and release the nucleic acids contained in the biologicalcompartments. This procedure is preferably performed at temperaturesabove room temperature, and more preferably, at temperatures between 70and 95° C. The mixture created when the cells are lysed is also referredto as the lysis mixture below. The incubation period is preferably from5 to 20 minutes, and more preferably, from 10 to 15 minutes. If thecells are lysed at room temperature or a temperature slightly above roomtemperature, it is especially preferable to then warm the lysis mixtureto higher temperatures such as 70° C. or, if the samples are potentiallyinfectious, to 95° C. The lysis can also be deactivated if it interfereswith the subsequent steps.

The lysis mixture is then cooled under conditions that depend on thepurpose for which the procedure provided by this invention is performed.If the nucleic acids are isolated on a solid phase, conditions areselected under which the nucleic acids can bind to the solid phase. Asuitable procedure for binding nucleic acids is the incubation ofreleased nucleic acids with glass surfaces in the presence of chaotropicsalts. A procedure of this nature is described in EP-A-0 389 063, forinstance. In this procedure, the nucleic acids are boundnon-specifically to the glass surface, while other components of thebiological compartments and the lysis reagent are not bound to the glasssurface, or are only bound insignificantly. The fluid that contains theremaining components is then preferably removed from the sampleprocessing vessel, e.g. via aspiration, while the glass surface canremain in the sample processing vessel with the nucleic acids bound toit. In a preferred construction, a solid phase in the form of a glassfiber fleece is placed in the sample processing vessel and incubatedwith the mixture. In this procedure, the nucleic acids are immobilizedon the glass fibers and can simply be removed from the sample processingvessel with the glass fiber fleece.

If the nucleic acids will be detected after their release, they arehybridized with a probe. This probe, as described above, is a moleculethat has a base sequence that is complementary to the nucleic acid to bedetected or a part thereof. In a preferred case, this is anoligonucleotide labelled with a group to be detected. The reactionmixture is therefore cooled under conditions in which the nucleic acidsto be detected hybridize with the nucleic acid probe. These temperaturesare known by the expert. In another construction of the procedure fordetection of nucleic acids, the nucleic acids to be detected hybridizewith a nucleic acid probe bound to a solid phase. In this procedure, theprobe can be used on any solid phase, such as microtiter plate cavitiesor the inside wall of the sample processing vessel, as long as it isseparated only from the rest of the reaction mixture. Procedures forimmobilizing nucleic acid probes, especially "capture" probes, are knownby the expert, e.g. from EP-A-0 523 557.

After the mixture is cooled, the nucleic acids to be isolated ordetected are then separated from the surrounding fluid that may stillcontain remains of the lysis mixture and reagents used to bind thenucleic acids to a solid phase. Depending on the type of solid phaseused, the solid phase can be filtered or removed from the sampleprocessing vessel, or the fluid can be removed from the sampleprocessing vessel by pipette.

The bound nucleic acids can then be unbound from the solid phase,detected directly in common procedures for detecting nucleic acidsequences known by the expert, or labelled.

Version B

In the initial step, the sample is pipetted into a sample processingvessel along with lysing reagent and glass-magnetic particles (beads)that can bind with the nucleic acids contained in the biologicalcompartments. The sample processing vessel is then closed and shaken. Itis necessary for the sample, lysis reagent and glass-magnetic particlesto be placed in the sample processing vessel. Neither the method bywhich they are placed in the vessel nor the sequence in which this takesplace is especially significant for the procedure provided by thisinvention. Preferably, however, the glass-magnetic particles arepipetted into the sample processing vessel in a suspension with a knownconcentration of glass-magnetic particles. The sample is pipetted intothe sample processing vessel either before or after the suspension ofglass-magnetic particles. It is essential that the sample,glass-magnetic particles and lysis reagent are shaken until mixedthoroughly.

Another step of the procedure provided by this invention is thesubsequent disintegration (lysis) of the biological compartments.Procedures for lysing biological compartments are known by the expert,as are the specific conditions for certain types of compartnents, e.g.cells. To lyse bacteria, for instance, a mixture of proteinase K isadded to the biological compartments and incubated for the amount oftime necessary to lyse or partially or completely decompose the cellwalls and release the nucleic acids contained in the biologicalcompartments. This procedure is preferably performed at temperaturesabove room temperature, and more preferably, at temperatures between 70and 95° C. The mixture created when the cells are lysed is also calledthe lysis mixture below. The incubation period is preferably from 5 to20 minutes, and more preferably, from 10 to 15 minutes.

If the cells are lysed at room temperature or a temperature slightlyabove room temperature, it is especially preferable to then warm thelysis mixture to higher temperatures such as 70° C. or, if the samplesare potentially infectious, to 95° C. The lysis can also be deactivatedif it interferes with the subsequent steps.

An important feature of the invention is the fact that the mixture inthe sample processing vessel is shaken during incubation. The mixturecan be shaken in intervals, or it can be shaking during the entireincubation period or only during certain periods. The mixture is shakenin order to sufficiently mix the biological compartments and themagnetic particles in the fluid, and especially to suspend or resuspendthe beads and accelerate diffusion. This reduces the amount of timerequired to bind the biological compartments to the magnetic particles.

The lysis mixture is then cooled under conditions that depend on thepurpose for which the procedure provided by this invention is performed.The nucleic acids released from the biological compartment should nowbind non-specifically to the surface of the glass-magnetic particles. Toimprove the binding characteristics, i-propanol or ethanol--depending onthe type of biological compartment involved--is added to the lysismixture after lysis. The sample processing vessel is then shaken to mixthe mixture further. The nucleic acids are now bound non-specifically tothe surface of the solid phase. Other components of the biologicalcompartment and lysis reagents do not adsorb to the glass surface, oronly insignficant components adsorb to the glass surface.

After the solid phase binding step is complete, the magnetic fields areactivated in order to deposit the glass-magnetic pigment with the boundnucleic adds on the inside surface of the sample processing vessel. Theremaining fluid is then removed from the vessel. In a preferredconstruction, however, in which the sample processing vessel has anopening at the bottom from which the fluid can exit, the fluid is simplyaspirated through this opening. This fluid removal method minimizes themechanical stress placed on the magnetic particles and thereby preventsthem from being removed from the vessel wall.

In a subsequent step, the glass-magnetic particles are resuspended in awash fluid. To accomplish this, the magnet is moved away from the vesselso that it no longer holds the magnetic particles against the vesselwall. As described above, it is also possible to move the vessel awayfrom the magnet. With regard for the invention described here, it hasbeen demonstrated that simply removing the magnet is not enough toresuspend the magnetic particles in the solution if the vessel is notalso shaken, and preferably simultaneously. This shaking motion isperformed by the mechanical shaker (30) and causes the magneticparticles to be distributed evenly within the second fluid. This secondfluid can be added to the sample processing vessel, e.g. via pipette,before or after the magnet is removed. This wash step can be repeated asneeded until the biological compartments have reached a sufficient levelof purity.

The bound nucleic acids can then be unbound from the solid phase,detected directly in common procedures for detecting nucleic acidsequences known by the expert, or labelled.

The procedure provided by this invention is therefore based on acombination of steps that make use of a receptacle (10) for holding oneor more sample processing vessels, a thermostat unit (20) to maintainthe sample processing vessels and the fluid in them at a constanttemperature, a mechanical shaker (3) to shake the sample processingvessels, and a unit (40) for separating and depositing the magneticparticles on a wall of each sample processing vessel using magneticforce. Surprisingly, these steps and units can be combined into a singlereaction block. A reaction block in this case refers to a device thatcomprises all or some of the units 10, 20, 30 and 40 coupled incoordinated fashion. This invention enables a process to be performed inone instrument that used to require numerous manual preparation steps.The reaction blocks provided by this invention have been demonstrated tobe especially effective. Procedures for the release and isolation ofnucleic acids can now be performed more quickly with this system thanbefore. The system also makes it possible to leave the nucleic acids inthe vessel during the steps described. This represents a considerableadvance over the state of the art in terms of time savings and avoidingcontamination. Suspensions were usually cooled previously by manuallyremoving a sample processing vessel from the instrument and immersing itin a cooling bath. This procedure has been demonstrated to beinsufficient for the future of routine diagnostic testing procedures.

FIG. 3 shows a procedure for isolating nucleic acids provided by thisinvention. This figure is referred to in the procedure described in theexample below. The sample vessel is located in a receptacle in unit 10.The sample vessel preferably has a stem (A20) that is molded to fitwithin the receptacle (e.g. it is conical in shape). The vessels shownin cross-section can be made simply from polypropylene using injectionmolding techniques.

A main advantage of the invention is the fact that the system can beadapted to a large extent for use with different sizes of magneticparticles. It is relatively flexible and can be used with the mostdiverse procedures.

The object of the invention is explained in greater detail using theexample below:

EXAMPLE 1

The basic principles of the procedure provided by this invention areknown by experts in nucleic acid diagnostics. Any technical details notlisted below can be found in "Molecular Cloning" by J. Sambrook et al.,CSH 1989.

A particular construction of the procedure for processing samplesolutions containing nucleic acids provided by this invention comprisesthe following working steps (see FIG. 3). In the initial step (I), asample fluid containing cells is incubated in a sample vessel (A) with amaterial to which the cells bind and from which nucleic acids will beextracted. This material can have binding characteristics specific forcell surfaces, e.g. antibodies against surface antigens are immobilizedon an absorber material (A16, not shown). Or, the material can havefilter characteristics (A15, not shown) that retain the cells when thefluid flows through the material, e.g. as it is being removed from thesample vessel. Conditions for immobilizing cells on surfaces are knownby the expert, e.g. from "Methods in Enzymology", Vol. 171,Biomembranes/Part R Transport Theory: Cell and Model Membranes, editedby Sidney Fleischer, Becca Fleischer, Department of Molecular Biology,Vanderbilt University, Nashville, Tenn., pages 444 ff or 581 ff.

During incubation, the sample vessel is preferably closed with a cap (B)to ensure active and passive protection from contamination.

In a subsequent step, the fluid is removed from the sample vessel whilethe cells containing the nucleic acids to be isolated remain in thesample vessel bound to the material. Since the cell-binding material isa particular material, retention is achieved by the fact that thematerial is magnetic (manufactured by Dynal, Oslo, Norway), and themagnet is moved towards the sample vessel from the outside. The fluidcan be aspirated through the outlet (A11) by creating a slight vacuum.To accomplish this, a valve is built into the outlet that opens when avacuum is created.

One or more wash steps are provided in order to remove remaining samplecomponents from the cells that may cause interference. In these steps, awash fluid is added to the sample vessel in which any contaminantspresent dissolve but which does not have a negative effect on the cellsbinding to the surface of the cell-binding material. Wash solutions ofthis nature are known by the expert, e.g. from cell separation protocolsor appropriate purification kit protocols for nucleic acids. Theybasically depend on the way the cells are bound to the material.

Once the last wash solution is aspirated from the sample vessel (A), thepurified, enriched cells are brought in contact with an appropriatelysis fluid to release the nucleic acids from the cells. The reagents ofthis lysis solution basically depend on the type of cells immobilized(Rolfs et al.: PCR, Clinical Diagnostics and Research, SpringerPublishers, 1992, pg. 84 ff). If the cells are bacterial, the lysissolution preferably contains proteinase K to decompose the cell wall. Ifdesired, the lysis can be encouraged with heating or cooling steps ormixing the reaction mixture by shaking the sample vessel. When lysis iscomplete, the nucleic acids to be isolated are freely available in thesolution.

The reaction vessel is also preferably closed with a cap during thelysis step, in order to prevent contamination from the environment. Whenlysis is complete, the cap is removed, preferably by using anappropriate mechanical device. A moulded article (C) is then insertedinto the sample vessel that contains a mixture of cellular decompositionproducts and nucleic acids, the external contour of which (C12) iscoordinated with the internal contour (A 17) of the sample vessel. Thismoulded article is hollow and closed with a filter (C 11) (porousmatrix) on the end situated towards the sample vessel and the reactionmixture. The moulded article (C) is preferably inserted using acomponent (B 10) that is suitable for closing the sample vessel. In thiscase, the moulded article is held by the cap (II) and inserted into thesample vessel when it is closed. During this process, the reactionmixture also enters the hollow space (C 14) of the moulded articlethrough the filter (C 11) (IV). The filter can prevent large particlesfrom entering the hollow space and, due to its nucleic acid-bindingcharacteristics, it binds nucleic acids even as the reaction mixturepasses through. A filter material containing glass fibers is selectedfor use in this case.

In a subsequent step, the remaining lysis reaction mixture is removedfrom the device formed by A and C by aspirating it through the outlet(All) in the sample vessel. This procedure also removes the solutionthat entered the hollow body (C14) of the moulded article, leaving aslittle filter residue in the filter as possible. The cap (B) is thenremoved, but the moulded article (C) remains in the sample vessel.

At the same time or immediately thereafter, an elution vessel (D) isprepared to receive the moulded article (C) (either in the systemprovided by this invention or outside of it). If this vessel has a cap,it is removed (VI). Preferably, an elution solution is added to theelution vessel before the moulded article (C) is inserted into theelution vessel (D), e.g. using a pipette. The composition of the elutionsolution is based on the type of nucleic acid binding with the materialin the filter (C). It contains reagents that cause the immobilizednucleic acids to elute, e.g. dissolve, from the material. The cap (B)that originally covered the elution vessel is placed on the samplevessel (A) with the moulded article (C) (VII).

To remove the moulded article (C) from the sample vessel (A), themoulded article (C) is removed along with the cap (B) (VIII). Thecombination of cap and moulded article is then inserted in the elutionvessel (IX). The moulded article (C) preferably contains a means (C13,not shown) for fixing the moulded article in the elution vessel (D) thatensures that the moulded article can only be removed from the vessel (D)if the moulded article (C) or vessel (D) is destroyed, or a force isused that is stronger than the force used to loosen the cap (B) from themoulded article (C). It is not intended for the moulded article to beremoved from the elution vessel.

While the moulded article (C) enters the elution vessel, the elutionsolution in the vessel enters the filter (C 11) and loosens theimmobilized nucleic acids from the solid matrix. Depending on thequantity of elution solution in the vessel, the filter is either justsaturated with the elution solution, or the elution solution--and theredissolved nucleic acids--enter the hollow space (C 14). To ensure thatthe nucleic acids are eluted as completely as possible, the innercontour of the elution vessel should fit as tightly against the externalcontour of the moulded article as possible.

In a subsequent step, the cap (B) is removed from the combination ofmoulded article (C) and elution vessel (D) (X). It is used to pick up astamp (E) (XI) and insert it into the hollow space of the mouldedarticle (C) (XII). The cap grips the stamp (E) from inside. The stamp ispressed against the filter (C 11) with such force that fluid from thefilter enters an internal space in the stamp through an opening on thepressing surface. This procedure is especially effective if the externalcontour of the pressing surface is coordinated with the inner contour ofthe moulded article (C), at least in the area intended for pressing. Thestamp (E) can preferably be fixed in this position, e.g. by snapping itinto place. Since the cap closes the device with this constructionrelatively tightly, the solution containing nucleic acids can be storedin this device.

To remove a desired quantity of nucleic acid solution, the cap can beremoved (XIII) and the desired quantity of solution can be removedthrough an opening in the internal space of the stamp, e.g. by pipette(XIV). The cap can then be placed back on the tube.

The sequence of steps of the procedure described is provided below.

    ______________________________________                                        Instrument       Operator                                                     ______________________________________                                        automated (program-controlled)                                                                 manual                                                       incubate         pipette                                                      aspirate         place tubes, glass fleece insert and                                          back-up container on the instrument                          separate (magnetic solid phase)                                               mix/resuspend                                                                 ______________________________________                                    

Manual working steps are shown in bold. Non-manual working steps orpartial sequences are called up by pressing a key, for instance.

In the table below, the sample vessel A is called a tube, the elutionvessel D is referred to as the back-up vessel, the moulded article C isreferred to as the glass fleece insert, and the stamp E is called thepress stamp.

    ______________________________________                                        Step                                                                          #    Action                    Time (s)                                       ______________________________________                                         1   Place tube #1-16 in the reaction module.                                  2   Pipette receptor (50-100 μl) and SA beads (50-                             100 μl) in tubes #1-16                                                 3   Pipette sample (1000 μl) in tubes #1-16                                4   Close tubes with cap (16 each)                                            5   Mix. Frequency = 30 Hz (parallel)                                                                       30 s                                            6   Incubation. 9 = 4° C. After incubation 9 = RT                                                    300-1200 s                                          (parallel)                                                                6*  Mix (if necessary) during incubation                                      7   Magnet ACTIVATED (in-parallel)                                                                          5 s                                             8   Aspirate waste (sequentially, 5 s)                                                                      80 s                                            9   Magnet DEACTIVATED (in parallel)                                                                        5 s                                            10   Open the cap on the tubes (16 each)                                      11   1st wash step:                                                                Pipette 500-1000 μl wash solufion (low molecular                           weight salt) into tubes #1-16                                            12   Resuspend. Frequency = 30 Hz (in parallel)                                                              5 s                                            13   Magnet ACTIVATED (in parallel)                                                                          5 s                                            14   Aspirate waste (sequentially, 5 s)                                                                      80 s                                           15   Magnet DEACTIVATED (in parallel)                                                                        5 s                                            16   2nd wash step:                                                                Pipette 500-1000 μl wash solution (low molecular                           weight salt) into tubes #1-16                                            17   Resuspend. Frequency = 30 Hz (in parallel)                                                              5 s                                            18   Magnet ACTIVATED (in parallel)                                                                          5 s                                            19   Aspirate waste (sequentially, 5 s)                                                                      80 s                                           20   Magnet DEACTIVATED (in parallel)                                                                        5 s                                            20.1 3rd wash step (optional):                                                     Pipette 500-1000 μl wash solution (low molecular                           weight salt) into tubes #1-16                                            20.2 Resuspend. Frequency = 30 Hz (in parallel)                                                              5 s                                            20.3 Magnet ACTIVATED (in parallel)                                                                          5 s                                            20.4 Aspirate waste (sequentially, 5 s)                                                                      80 s                                           20.5 Magnet DEACTIVATED (in parallel)                                                                        5 s                                            21   Pipette lysis mix, reagent 1 + 2 (400 μl)                                  guanidinium hydrochloride or guanidinium                                      rhodanide and proteinase K (25 μl) in tubes #1-16                     22   Close the tubes with the caps (16 each)                                  23   Resuspend. Frequency = 30 Hz (in parallel)                                                              5 s                                            24   Incubation: 9 = 70° C.                                                                           600 s                                               [Optional: Incubation: 9 = 95° C. with potentially                                               900 s                                               infectious samples]                                                      25   Incubation: 9 = RT        300 s                                          26   Open the caps on the tubes (16 each)                                     27   Pipette 200 μl ethanol (isopropanol) into tubes #1-                        16                                                                       28   Close the caps bn the tubes (16 each)                                    29   Mix. Frequency = 30 Hz (in parallel)                                     30   Open the caps on the tubes (16 each)                                     31   Place glass fleece insert in tube #1 into tubes #1-16                    32   Aspirate waste (sequentially, 5 s)                                                                      80 s                                           33   Pipette 500 μl wash solution (chaotropic salt/                             ethanol) into tubes #1-16                                                34   Aspirate waste (sequentially, 5 s)                                                                      80 s                                           35   Pipette 500 μl wash solution (chaotropic salt/                             ethanol) into tubes #1-16                                                36   Aspirate waste (sequentially, 5 s)                                                                      80 s                                           37   Place back-up vessel on the instrument (#1-16)                           38   Pipette elution volume into the back-up vessel                                (100-200 μl) into tubes #1-16                                         39   Transfer glass fleece insert from tube #1 to the                              back-up vessel (#1-16)                                                   40   Insert press stamp (#1-16) into the back-up vessel -                          Elution                                                                  41   Close the back-up vessel (16 caps)                                       42   Remove tubes #1-16 from the RM - waste                                   ______________________________________                                    

If desired, the suction tubes and the cavities can be rinsed and,therefore, cleaned, with a cleaning fluid (before or after the procedureis performed and after the sample vessels have been removed).

Another construction of a system for the release and isolation ofnucleic acids is shown in FIGS. 4 through 8.

FIG. 4: Perspective drawing of the system

FIG. 5: Phantom drawing of the system

FIG. 6: Receptacle for sample processing vessels

FIG. 7: Cross-section through a receptacle for a sample vessel

FIG. 8: Pushing device with magnets

The system shown in FIG. 4 has 4 receptacles (100) for sample vessels.The receptacles (100) are fixed to a carrier (101). Moving the carrier(101) moves and shakes the 4 receptacles (110) in unison. The phantomdrawing (FIG. 5) illustrates in greater detail how the movement of thecarrier (101) takes place. The carrier has a circular recess (102) oneach of its 2 ends in which a rod (103) is situated. The rod (103) issituated on the axis of a motor (104) in an eccentric position. When themotor axis turns, therefore, the carrier is shifted along a plane. Thephantom drawing (FIG. 5) also illustrates clearly that the receptacle(100) has cooling fins (105). A stream of air is blown through thecooling ribs by means of ventilating fans (106) located on the base ofthe system to cool the system.

A receptacle (100) is shown in greater detail in FIG. 6. The receptaclehas 6 separate cavities (107) to receive sample processing vessels. Thecavities (107) are connected with the peltier element (109) by means ofa framework (108) in a thermally conductive fashion. The peltierelement, in turn, is connected with the cooling fins (105). The frame(108) contains temperature sensors and a resistance heating device towarm the cavities. The frame (108) is mounted on a peltier element (109)that exchanges heat with the cooling fins (105). The sample processingvessels are warmed by the heating elements located in the frame. Thecavities (107) are cooled by the peltier element (109) in order to coolthe sample processing vessels. The heat emitted by the peltier elementon the opposite side is eliminated through the cooling fins (105).

FIG. 7 shows a cross-section of one cavity (107). The cavity consists ofa cylindrical space in which a sample vessel (110) is located. Thesample vessel and cylindrical space are designed in such a way that thewalls touch each other, to promote efficient heat transfer. Preferably,the sample vessel and cavity are both slightly conical, i.e. they tapertowards the bottom, so that a snug fit is achieved. The conicity ispreferably between 0.5 and 1°. An insert is situated in the lowerportion of the cavity that has a recess (112) on its underside intowhich a hose connection can be screwed. On the upper ends the insert(112) has an opening into which the tapered tip of a sample vessel (110)can be inserted. To achieve a tight connection between this opening andthe tapered tip of the sample vessel, a sealing ring (113) in the shapeof an O-ring that surrounds the tapered tip of the sample vessel islocated in this position. To remove fluid from the sample vessel (110),a vacuum is created in the sample vessel (110) by means of a tube thatis connected to the opening (112).

When the system is operated as provided by this invention, a magneticfield can be applied to the sample vessels (110) while they are situatedin the cavities (107), to hold magnetic particles. The magnet assemblyshown in FIG. 8 is moved towards the cavities (107) for this purpose.

FIG. 8 shows a moveable assembly of 4×6 magnets (114), corresponding tothe 4 receptacles (110) with 6 cavities (107) each. The assembly shownin FIG. 8 has a rail (115) on which 4 units of 6 magnets each aremounted. The rail (115) is fixed to the carrier (116) which, in turn,holds a motor (117). A gearwheel (not shown) is mounted to the axis ofthe motor that grips the teeth (not shown) of a gear rack (118). Thecarrier (116) is arranged on cylinders (120) in such a fashion that itcan be pushed along by linear ball bearings (119). In the position inwhich magnetic particles are deposited, the front surfaces of themagnets (114) are located directly against the flat sides (121) of thecavities (107). To move the magnets away from the cavities, the motor(117) is activated and the carrier, including the rail (115) is moved inlinear fashion.

    ______________________________________                                        Reference Drawing List                                                        ______________________________________                                        A         Sample Vessel                                                           10    Inlet                                                                   11    Outlet                                                                  17    Internal contour                                                        19    External contour                                                        20    Stem base                                                               22    Element to which additional functional elements can be                        attached                                                            B         Cap                                                                     10    Component for closing sample vessel A                                   11    Component for gripping the moulded article C                        C         Moulded Article                                                         11    Porous matrix                                                           12    External contour                                                        13    Means for flxing the moulded article in the elution vessel              14    Hollow body                                                             15    Means for attaching a cap                                               16    Internal contour                                                        17    Means for fixing a stamp E into position, all the way around            18    Stem base, can be broken off                                            19    Edge                                                                D         Elution Vessel                                                          12    Snap-in notch                                                       E         Stamp                                                                   10    Pressing surface                                                        11    External contour                                                        12    Internal space                                                          13    Openings in the pressing surface                                        14    Opening for removing contents                                           15    Seal                                                                    16    Snap-in ring                                                            17    Recess                                                              ______________________________________                                    

    ______________________________________                                        Instrument                                                                    ______________________________________                                        1    Frame                                                                    10   Receptacle for sample vessels                                            11   Vibration absorber                                                       12   Cavity                                                                   13   Base                                                                     14   Inlet to heat and cool A                                                 20   Thermostat unit to maintain sample vessels at a constant                      temperature                                                              21   Duct for cooling/heating                                                 30   Mechanical shaker for sample vessels                                     40   Separator for separating magnetic particles using magnetic force         41   Axis for turning magnet segments                                         42   Magnet segments                                                          50   Vacuum pump/pump unit                                                    51   (Vacuum) tube                                                            100  Receptacle for sample vessels                                            101  Carrier                                                                  102  Circular recess                                                          103  Rod                                                                      104  Motor                                                                    105  Cooling fins                                                             106  Ventilator                                                               107  Cavity                                                                   108  Frame                                                                    109  Peltier element                                                          110  Sample vessel                                                            111  Insert                                                                   112  Opening                                                                  113  Sealing ring                                                             114  Magnet                                                                   115  Rail                                                                     116  Carrier                                                                  117  Motor                                                                    118  Gear rod                                                                 119  Linear ball bearing                                                      120  Cylinder                                                                 ______________________________________                                    

What is claimed is:
 1. A system, comprisinga sample processing vesselhaving an outlet opening and being capable of containing fluid andbiological compartments; a receptacle defining at least one cavity whichholds therein the sample processing vessel, wherein said receptaclecomprises a hose connection for connecting a hose to the outlet opening;a plurality of magnetic particles for introducing into the sampleprocessing vessel, wherein said magnetic particles are capable ofbinding with the biological compartments; a thermostat unit configuredto maintain the sample processing vessel and its contents at anessentially constant temperature; a mechanical shaker configured toshake the sample processing vessel; and a magnetic means formagnetically attracting said magnetic particles to an inner wall of thesample processing vessel, wherein said receptacle, said thermostat unitand said magnetic means are integrated into a reaction block.
 2. Thesystem of claim 1, wherein said sample processing vessel containsbiological compartments.
 3. The system of claim 1, further comprising ahose which is connected to said hose connection.
 4. The system of claim1, further comprising a hose, wherein one end of said hose is connectedto said hose connection.
 5. The system of claim 1, wherein said magneticmeans and said receptacle are configured to move towards or away fromone another.
 6. The system of claim 1, wherein said receptacle definesat least two cavities, wherein each cavity is configured to hold thereina sample processing vessel.
 7. The system of claim 6, further comprisinganother sample processing vessel, wherein each of the sample processingvessels is held in a corresponding cavity.
 8. The system of claim 6,wherein the cavities are arranged in an essentially linear format. 9.The system of claim 1, wherein said mechanical shaker comprises aneccentric drive.
 10. The system of claim 1, wherein said thermostat unitcomprises a thermally conductive metal block which is integrated intosaid receptacle, and the at least one cavity is configured to snuglyhold therein the sample processing vessel such that said metal block iscapable of efficiently transferring heat between said metal block andthe sample processing vessel.
 11. The system of claim 1, wherein saidthermostat unit comprises a resistance heating unit and a peltierelement.
 12. The system of claim 1, wherein said sample processingvessel and the at least one cavity have essentially matching conicity.13. The system of claim 1, wherein said thermostat unit regulates thetemperature by means of a fuzzy regulator.
 14. The system of claim 1,wherein said thermostat unit regulates the temperature by means of aproportion integral differential time regulator.
 15. The system of claim1, wherein said thermostat unit comprises a block through which athermostating medium flows.
 16. The system of claim 1, wherein saidmagnetic particles comprise ferromagnetic particles.
 17. The system ofclaim 1, further comprising a pump unit which is connected to said hoseconnection, wherein said pump unit is configured to remove the fluidfrom the outlet opening through the hose.
 18. A system, comprisingasample processing vessel having an outlet opening and being capable ofcontaining fluid and biological compartments; a receptacle defining atleast one cavity which holds therein the sample processing vessel,wherein said receptacle comprises a hose connection for connecting ahose to the outlet opening; a plurality of magnetic particles forintroducing into the sample processing vessel, wherein the magneticparticles are capable of binding with the biological compartments; athermostat means for maintaining the sample processing vessel and itscontents at an essentially constant temperature; a shaking means forshaking the sample processing vessel; and a magnetic means formagnetically attracting said magnetic particles to an inner wall of thesample processing vessel, wherein said receptacle, said thermostat meansand said magnetic means are integrated into a reaction block.
 19. Thesystem of claim 18, further comprising a pump unit which is connected tosaid hose connection, wherein said pump unit is configured to remove thefluid from the outlet opening through the hose.